The complement system is capable of tissue and cell destruction and is therefore a major element of the defense system against invasion by foreign tissue. However, control of this system is necessary in order to prevent destruction of autologous cells. A large number of proteins which are involved in control of the complement cascade have been described.
Most relevant to the present invention is the group which controls the C3 convertase stage of the cascade and binds to fragments of either C3 or C4 or both. This group includes serum proteins such as C4-binding protein and factor H and membrane proteins such as C3b receptor, C3d/Epstein-Barr virus receptor, decay-accelerating factor (DAF), and the protein of the invention, membrane factor protein (MCP). Reviews of these various factors and their role in complement cascade regulation can be found in Holers, B. M., et. al., Immunol Today (1985) 6:188; Ross, G. D., et. al., Adv Immunol (1985) 37:217; Atkinson, J. P., et. al., Immunol Today (1987) 8:212.
Much is known concerning these regulatory proteins, except for MCP. They are each composed of multiple repeats of an approximately 60-amino acid consensus sequence composed of conserved cys, pro, gly, trp, leu/ile/val, and tyr/phe residues (Reid, K., et al., Immunol Today (1986) 7:230). The genes encoding these proteins have been localized to the long arm of human chromosome 1, band lq32 and form a multigene family designated the regulator of complement activation (RCA) gene cluster. As will be shown below, MCP is also a member of this family.
A member of this family particularly related to the MCP of the invention is the decay-accelerating factor (DAF) which was identified on human platelets by Yu, G. H., et. al., J Clin Invest (1986) 78:494-501. DAF is present on virtually all peripheral blood cells, including erythrocytes, granulocytes, T and B lymphocytes, monocytes, and platelets; in addition, soluble forms of DAF have been found in extracellular fluids and tissue culture supernatants. The gene encoding DAF has been cloned and sequenced (by Medof, M. E., et. al., Proc Natl Acad Sci USA (1987) 84:2007-2011; and by Caras, I. W., et. al., Science (1987) 238:1280-1283). It has been shown that the membrane and soluble secreted forms of DAF result from differential splicing of the mRNA encoding these proteins with the soluble form having a longer C-terminus, but a C-terminus which lacks the membrane binding region associated with the membrane DAF, as described in PCT application WO89/01041.
MCP was initially identified by iC3/C3b affinity chromatography on surface-labeled peripheral blood cells and designated gp 45-70 to describe the range of M.sub.r obtained on SDS-PAGE (Cole, J. L., et. al., Proc Natl Acad Sci USA (1985) 82:859). MCP was partially purified from the human mononuclear cell lines and shown to have a cofactor activity but no decay accelerating function (Seya, T. J., et. al., J Exp Med (1986) 163:837).
Complement Components. Human C1s (Takahashi, et. al. "The NH.sub.2 -terminal sequences of a subunit of the first component of human complement C1s, and its activated form, C1s", FEBS 50:330 (1975)), C4 (Nagasawa and Stroud "Purification and characterization of a macromolecular weight cofactor for C3b-inactivator, C4bC3bINA-Cofactor, of human plasma", Mol. Immunol. 17:1365 (1980)), C2 (Nagasawa and Stroud "Cleavage of C2 by C1s into the antigenically distinct fragments C2a and C2b. Demonstration of binding of C2b to C4b" Proc. Natl. Acad. Sci. USA. 74:2998 (1977)), D (Volanakis, et. al., "Human factor D of the alternative complement pathway: Purification and characterization", J. Immunol. 119:337 (1977)), and B (Seya, et. al., "Generation of C3d.g and C3d by urokinase-treated plasma in association with fibrinolysis", Complement 2:165 (1985)), were purified as described. These components were assessed by gel criteria for purity and by fluid-phase classical and alternative pathway assay systems to be certain that they were free of H, C4bp, and C3 (Seya, et. al. "Purification and functional analysis of the polymorphic variants of the C3b/C4b receptor (CR1) and comparison to H. C4b-binding protein (C4bp) and decay accelerating factor (DAF)", J. Immunol. 135:2661 (1985)). C3 was purified as previously described by Nagasawa and Stroud, "Mechanism of action of the C3b inactivator: Requirement for a high molecular weight cofactor (C3b-C4b INA Cofactor) and production of a new C3b derivative (C3b')", Immunochemistry 14:749 (1977). In the assays of cofactor activity of I-mediated cleavage, C3 was further purified by passage through a column of anti-H antibody conjugated to Sepharose and rechromatography on DEAE-Sephacel to eliminate C4bp (Seya, et. al., J. Immunol. 1985). C3 (H.sub.2 O) and methylamine (MA)-treated C3 (C3.sub.MA) were prepared as previously noted (Seya, et. al., J. Immunol. 1985). C3(H.sub.2 O) and C3.sub.MA are hemolytically inactive C3 produced by repetitive freeze-thaw cycles and MA treatment, respectively. C4.sub.MA is hemolytically inactive C4 produced by MA treatment. The conversion of C3 to C3(H.sub.2 O) was confirmed by the heat-induced autocleavage reaction (Seya and Nagasawa "Limited proteolysis of the third component of human complement" C3, by heat treatment J. Biochem. 89:659 (1981)). Human I (Nagasawa and Stroud, "Cleavage of C4b by C3b inactivator: Purification of a nicked form of C4b, C4b', as an intermediate cleavage product of C4b by C3b inactivator" J. Immunol. 125:578 (1980)), C4bp (Nagasawa, et. al., "Limited chymotryptic cleavage of human C4b binding protein: Isolation of a carbohydrate-containing core domain and an active fragment" J. Biochem. 92:1329 (1982)), and H (Seya and Nagasawa, "Limited proteolysis of complement protein C3b by regulatory enzyme C3b inactivator: Isolation and characterization of a biologically active fragment", C3d.g. J. Biochem. 97:373 (1985)) were purified as described. H was depleted of C3 by rechromatography on QAE-Sephadex. C4bp contained no detectable C3 or H as assessed by radioimmunoassay (Seya, et. al., J. Immunol. 1985). All of these components were dialyzed against PBS, pH 7.2, and stored in aliquots at -70.degree. C.
MCP is absent from erythrocytes, but present as a membrane-bound protein on human T and B lymphocytes, granulocytes, monocytes, platelets, endothelial cells, epithelial cells, and fibroblasts; on most of these cells it occurs in polymorphic forms of molecular weight 63 kd and 58 kd, as determined by SDS-PAGE. These appear to result from a two allelic system encoding MCP (Ballard, L., et. al., J. Immunol (1987) 138:3850-3855). The MCP found by immunoprecipitation on the membranes of granulocytes appears, however, not to exhibit this polymorphism (Seya, T., et. al., Eur J Immunol (1988) 18:1289-1294). The occurrence of MCP on a wide range of host cells is consistent with a role in protecting host cells from damage by complement (Seya, T. L. et. al., Complement (1987) 4:225).
The previously purified MCP has been utilized to prepare a polyclonal rabbit antiserum monospecific for this protein. The antisera were raised in rats by repetitive injections of MCP purified as described by Seya, T., et. al., J Exp Med (1986) (supra), in complete Freund's adjuvant. These antisera have been used to identify MCP in extracts from various membranes.
The present invention provides a more highly purified form of this protein and the capacity to produce it recombinantly, thus providing practical quantities for therapeutic use.